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The importance of various factors relating to the morphological quality of corneas used for PKP by the Warsaw Eye Bank from 1996 to 2002.

To examine the effect of various factors such as: donor's age, cause of death, time between death and preservation and duration of preservation on the morphological quality of corneas used for Penetrating Keratoplasty (PKP). Our purpose was to assess the role of the above factors influencing the corneal overall rating and endothelial cell density. The present data regarding the donor's age and time between death and preservation of corneas obtained from eye banks belonging to the European Eye Bank Association and Polish eye banks were compared. Corneoscleral buttons and data concerning donors were obtained by eye bank technicians and collected in Warsaw Eye Bank in the years 1996-2002. The quality of the corneas recovered was evaluated by means of slit lamp Nikon NS-IV and specular microscope KONAN. Cardiorespiratory failure and cardiac arrest were the most frequent cause of the donors' death. In many cases donors with confirmed brain death who were given the corneas, were also donors of internal organs like heart, kidneys, pancreas and liver. The number of donors with disseminated neoplasm disease--complete disadvantage for removing corneas increased during the eye bank activity. The epithelial and endothelial layers sometimes underwent mechanical trauma in the group of donors because of sudden death. From 1996 to 2002 more than 50% of the donors were over 60 years of age. There were many problems with receiving corneas from younger donors. The overall rating tissues which were obtained in a very short time after death (to 5 hours) was higher (Excellent and Very Good) compared with corneas removed 8-12 hours after the donor's death. The increasing percentage of endothelial cell loss was observed in all the corneas after about 7 days of preservation in the medium. The mean endothelial cell density slightly decreased with donor's age, but its suggested range of the factors possibility of finding the corneas with high number of endothelial cell density both in younger and older donors. The average time from death to preservation was similar in eye banks preserving corneas at +4 degrees C (8-10 hours) compared with eye banks using mainly the organ culture method. The rating of the morphological state of corneas suitable for PKP depends on the time between death and preservation, donor's age, cause of death and time of preservation of corneas. Corneas obtained shortly after the donor's death showed a higher endothelial cell density and better overall rating than those removed after relatively longer period after the donor's death. An increasing percentage of endothelial cell loss was observed after 7 days of preservation independent of other factors.

Adolescent↗

Phosphatic metabolites of the intact cornea by phosphorus-31 nuclear magnetic resonance.

The principal low molecular weight phosphatic metabolites of the intact cornea were identified and quantitated nondestructively by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy. As part of this analytical procedure, the intracorneal pH was approximated from the resonance shift position of inorganic orthophosphate. In addition the metabolic and pH stability of incubated corneas at 37 C in MK medium was evaluated during an 8-hr time course and compared to similar dynamic analyses performed on corneas with denuded endothelium. Perchloric acid extracts prepared from these same corneas were analyzed by P-31 NMR to verify the metabolite peak assignments and to quantitate the concentrations of minor corneal metabolites. The concentrations of phosphatic metabolites of the cornea, including three previously unidentified phosphorus-containing substances, were determined for freshly excised corneas. The initial corneal spectroscopic profile was not altered by removal of the endothelium. At 37 C the MK media-incubated intact whole corneas experienced a time-dependent decline in ATP levels with a concomitant rise in inorganic orthophosphate; however, the tissue levels of the other principal phosphatic metabolites were not altered by prolonged incubation. In contrast, removal of the endothelial layer of the cornea-induced progressive metabolic deterioration of intact corneas characterized, most prominantly, by time-dependent declines in ATP and glycerol 3-phosphorylcholine levels and concomitant increases in ADP and inorganic orthophosphate levels relative to intact whole corneas. This study has established the feasibility of monitoring the metabolic status of intact rabbit corneas nondestructively and noninvasively. As such, P-31 NMR spectroscopy offers a promising method that may enable analysis of the metabolic viability of intact human donor corneas to provide a basis for selecting donor corneas for transplantation.

Adenosine Triphosphate↗

Altered expression of growth factors and cytokines in keratoconus, bullous keratopathy and diabetic human corneas.

The purpose of this study was to identify the growth factors and cytokines present in normal and diseased corneas. Total RNA was isolated from normal and diseased corneas. cDNA was synthesized from individual corneas and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with primers to IL-1alpha, 1IL-8, PDGF-B, BMP-2, BMP-4, IGF-I, TGF-beta2, FGF-2, and VEGF. After normalization to beta2-microglobulin, several factors were identified that were significantly different from normal. Antibodies to IGF-I, BMP-2, VEGF and TGF-beta2 were used for immunohistochemistry. A total of 93 corneas were used for this study including 31 normal, 20 keratoconus, 19 bullous keratopathy (pseudophakic and aphakic, PBK/ABK), and 23 diabetic corneas. The VEGF RNA levels were significantly decreased in the keratoconus and PBK/ABK corneas but increased in the diabetic corneas. BMP-2 gene expression was lower than normal in the PBK/ABK and diabetic corneas. IGF-I and BMP-4 RNA levels were increased in PBK/ABK. In the immunohistochemical studies, the protein patterns paralleled those found at the mRNA level. The only exception was IGF-I in diabetic corneas that showed increased staining in the epithelium and its basement membrane without a significant increase in mRNA levels. TGF-beta2 mRNA and protein levels were similar to normal in all diseased corneas. Thus, no alterations in the tested growth factors/cytokines were unique to keratoconus corneas. In contrast, PBK/ABK corneas had specific significant elevations of BMP-4 and IGF-I. Diabetic corneas were unique in their increased VEGF mRNA levels. These data suggest that while some growth factor/cytokine alterations are non-specific and can be found in multiple corneal diseases, there are others that are unique to that disease.

Adult↗

Relationships between eye factors and lens-forming transformations in the cornea and pericorneal epidermis of larval Xenopus laevis.

Larval Xenopus laevis at stage 56 (Nieuwkoop and Faber, '56) were subjected to various types of lentectomy: (1) simple lentectomy, from the pupillary space after incision of outer and inner cornea; (2) lentectomy from the dorsal region of the eye; (3) lentectomy from the dorsal region of the eye and simultaneous incision of the outer cornea; (4) lentectomy from the dorsal region of the eye and simultaneous incision of the outer and inner cornea. The results obtained show that the outer cornea underwent lens-forming transformations only when the inner cornea had been incised, thus permitting outer cornea (Experiments I-IV). No lens regeneration occurred when the inner cornea was left intact (Experiments II, III). It was concluded that the factor(s) allowing the lens-forming transformations of the outer cornea is not an aspecific nutritional factor(s) but a more specific factor(s) that cannot reach the outer cornea when the inner cornea is intact. Therefore, the absence of the lens and sufficient nutrient available to the outer cornea are not enough to allow lens regeneration from the outer cornea. When lens removal was carried out through the dorsal part of the eye (Experiments III-IV) the lens regenerated from the pericorneal epidermis of this region in a large number of cases.

Animals↗

Properties of corneas reconstructed with cultured human corneal endothelial cells and human corneal stroma.

PURPOSE: To examine the properties of corneas tissue-engineered with cultured human corneal endothelial cells (HCEC) and human corneal stroma. METHODS: Primary HCEC cultures were established from endothelial cell layer explants and propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. A cell suspension of HCEC at the fifth passage was transferred onto human corneal stroma deprived of endothelial cells, and the corneas were gently centrifuged to enhance cell attachment. The cell density of the tissue-engineered corneas was examined after staining with alizarin red and trypan blue. The tissue-engineered corneas were histologically examined by light and electron microscopy. The pump function of the tissue-engineered corneas was measured using an Ussing chamber. RESULTS: The mean endothelial cell density of four tissue-engineered corneas was 2380 +/- 264 cells/mm(2) (mean +/- SD). HCEC on the tissue-engineered corneas had a morphology similar to HCEC in vivo. The pump function parameters of the tissue-engineered corneas were 55%-75% of those of normal corneas. CONCLUSIONS: HCEC on the tissue-engineered corneas have morphology and cellular density similar to HCEC in vivo, whereas the pump function of the tissue-engineered corneas was lower than in normal corneas.

Adult↗

Age-related changes in superoxide dismutase, glutathione peroxidase, catalase and xanthine oxidoreductase/xanthine oxidase activities in the rabbit cornea.

The activities of superoxide dismutase, glutathione peroxidase (GPX) and catalase--the enzymatic scavengers of reactive oxygen species and the activities of xanthine oxidoreductase and xanthine oxidase, an enzyme known to generate reactive oxygen species, were studied in the corneas of normal rabbit eyes of various ages (1 month--young eyes; 4-9.5 months--young adult eyes; 2.0-2.75 years--middle aged eyes; 3.0-5.0 years--aged eyes). The activities of GPX, superoxide dismutase, xanthine oxidoreductase and xanthine oxidase were investigated biochemically in the scraped corneal epithelium. Catalase activity was detected histochemically in the corneal epithelium and endothelium. The results show that young corneas revealed lower activities of all the antioxidant enzymes investigated than did young adult corneas, in which enzymatic activities reached their maximum. In middle-aged corneas, GPX and catalase activities remained approximately at the same levels as seen in young adult corneas, whereas superoxide dismutase activity was decreased. In aged corneas, the activities of all antioxidant enzymes were dramatically decreased or even lost (catalase activity in the corneal endothelium). In contrast, xanthine oxidoreductase activity only slightly decreased with age and the xanthine oxidase proportion of total xanthine oxidoreductase remained unchanged. GPX, superoxide dismutase and catalase are important antioxidant enzymes protecting the cornea against the oxidative damage. Because the activities of these enzymes are lower in young animals and greatly reduced in aged animals, it is suggested that young and particularly aged corneas might be more susceptible to oxidative stress than are young adult corneas. This presumption is supported by the fact that the activities of prooxidant enzymes (xanthine oxidoreductase/xanthine oxidase) are only slightly decreased in aged corneas as compared to young adult corneas so that some imbalance between antioxidant and prooxidant enzymes exists already in the normal aged corneas.

Aging↗

Wound healing in rabbit corneas after photorefractive keratectomy and laser in situ keratomileusis.

PURPOSE: To compare the wound-healing process in the rabbit cornea after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) with the same refractive correction. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: Adult albino rabbits (N = 24) were used. One eye of each animal had PRK or LASIK with the same refractive correction. Each animal was killed after an interval of up to 6 months. The expression pattern of corneal stromal injury-related molecules with the 2 treatments were compared. Paraffin sections of the cornea were processed immunohistochemically for alpha-smooth muscle actin (alpha-SMA), collagen type IV [alpha1(IV)](2),alpha2(IV), and heat shock protein (HSP) 47 as well as other HSPs. Sections were also examined after hematoxylin and eosin or periodic acid-Schiff staining. RESULTS: Hematoxylin and eosin staining showed the central epithelium to be thick in PRK-treated corneas. The thick epithelium was restricted to the area around the corneal flap edge adhesion in LASIK-treated corneas at 3 months. Periodic acid-Schiff staining showed an absence of or interruption in the epithelial basement membrane in PRK-treated corneas for up to 6 months. Heat shock protein 47 was detected in keratocytes on day 3 but not after that in PRK-treated corneas. There was no difference in the expression of other HSPs. Alpha-smooth muscle actin was expressed in keratocytes repopulated in the central anterior cornea of PRK-treated corneas at 28 days. Keratocytes with immunoreactivity for these 2 proteins were not seen in LASIK-treated corneas. Collagen IV [alpha1(IV)](2),alpha2(IV) was not detected in either group of corneas. The central epithelium became transiently thicker in PRK-treated corneas. CONCLUSION: Keratocyte responses to laser stromal ablation were more marked in corneas treated with PRK than in those treated with LASIK.

Actins↗

Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas.

PURPOSE: Determine the tissue distribution patterns for tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3), gelatinase A and gelatinase B in normal and pathologic corneas. METHODS: Corneas were examined by immunohistochemistry, using antibodies to TIMP-1, TIMP-2, TIMP-3, gelatinase A or gelatinase B. RESULTS: In normal corneas, TIMP-1 antibody stained the epithelium and endothelium. TIMP-2 and TIMP-3 stained the epithelium, keratocytes and endothelium. Gelatinase A staining was weak and restricted to the epithelial cells. Radial keratotomy scars showed increased staining for TIMP-1 and TIMP-2 around the epithelial cell plug and along the incision. Bullous keratopathy corneas showed TIMP staining patterns similar to normal corneas and increased gelatinase A staining in regions of subepithelial fibrosis. Stromal scars of keratoconus corneas also had increased staining with TIMP-1 and TIMP-2 antibodies. In many keratoconus corneas, the TIMP-3 staining pattern was similar to normal corneas. However, in some keratoconus corneas, when Bowman's layer was missing, the stroma beneath was completely devoid of TIMP-3 antibody staining. No gelatinase B was seen in either the normal or diseased corneas. CONCLUSION: These data suggest that TIMP-1 and TIMP-2 are important for scar formation and corneal remodeling, since they were found in increased amounts at radial keratotomy incision sites and keratoconus scars. The significance of the focal stromal defects in TIMP-3 staining, associated with absence of Bowman's layer on keratoconus corneas, needs to be elucidated. At the stages of disease examined in this study, gelatinase B may not play a significant role in these pathological processes, since it was not seen in any of the corneas examined.

Base Sequence↗

Acute calcification in alkali-injured rabbit cornea treated with synthetic inhibitor of metalloproteinases (SIMP).

PURPOSE: The aim of this study was to evaluate the ultrastructure of acute calcification observed in alkali-injured rabbit corneas treated with synthetic inhibitor of metalloproteinases (SIMP). METHODS: A 12-mm alkali burn was made on the right corneas of 16 rabbits. Eight eyes were treated with topical phosphate-buffered SIMP, and eight control eyes were given the buffer only. Six corneas (five in the SIMP group and one in the control group) developed chalky white opacification in the central area of the cornea within 6-30 days. The corneas were excised for the following examinations: (a) light microscopy, (b) energy-dispersive x-ray analysis, (c) electron microscopy, and (d) high-angle x-ray diffraction. RESULTS: The results confirmed that there was stromal calcification in these corneas, as shown by positive van Kossa staining, needle-shaped calcium crystal aggregates, and increased calcium in the opaque areas of the corneas. Proteoglycans in the corneal stroma of both the SIMP group and the control group significantly decreased after alkali burns. Very few proteoglycan filaments could be identified in the calcified areas of the stroma. X-ray diffraction reflections with a Bragg spacing of 3.44 +/- 0.04 A were observed from all the calcified corneas treated with SIMP. The intermolecular spacings of collagen fibrils in the calcified stroma (1.24 +/- 0.34 nm) were significantly lower than those in the noncalcified stroma (1.64 +/- 0.04 nm) and the control corneas (1.64 +/- 0.02 nm). The results indicate that the collagen molecules within the fibrils of the calcified stroma are packed more closely than normal. CONCLUSION: The comparison of the x-ray diffraction patterns from the calcified corneas and chicken bone suggests that the calcium deposits in these corneas are in a form similar to those found in bone. The higher rate and more extensive calcification in the SIMP-treated corneas may result from the inhibitory action of SIMP on debris processing, but the mechanism is unknown.

Acute Disease↗

Changes of MK medium during storage of human cornea.

By comparing the composition of McCarey-Kaufman (MK) medium before and after corneal storage we attempted to identify specific physiological changes in the medium as predictors of tissue damage. We also tried to determine if hydrocortisone (a lysosomal membrane stabiliser) added to the medium could reduce tissue damage during storage. Corneas (human and rabbit) were stored in the MK medium with and without hydrocortisone for 4 days at 4 degrees C. The water and nitrogen contents of the stored cornea were compared with those of the fresh cornea. The medium was analysed before and after corneal storage to determine the concentrations of glucose, protein, and amino acids as well as pH and osmolarity. Scanning electron microscopy (SEM) was used to estimate the degree of the corneal endothelial cell damage. The nitrogen contents and dry weights of the steroid treated and untreated stored corneas were similar to those of the fresh unstored cornea. The steroid treated cornea contained a lesser amount of water than the untreated cornea. The cornea stored in medium without steroid took up a greater amount of glucose from the medium than the cornea stored in medium with steroid. As compared with their concentrations in the fresh unused medium the concentrations of leucine, lysine, and glycine were lower and that of glutamic acid was higher in both the media used for corneal storage. However, the steroid treated storage medium as compared with the untreated storage medium had a greater reduction in the lowering of leucine, lysine, and glycine, and a lesser reduction in the increase of glutamic acid. Steroid treated medium also had a lesser amount of protein released from the stored cornea. Changes in the pH and osmolarity of the media before and after corneal storage were not remarkable. SEM showed that the endothelial cells of the cornea stored in the medium containing steroid were less damaged than those of the cornea stored in the medium without steroid.

Amino Acids↗

[Comparing cryopreserved with fresh corneas on clinical application in penetrating keratoplasty].

PURPOSE: To evaluate the role of cryopreserved corneas on penetrating keratoplasty (PKP) by comparing them with fresh corneas on clinical application. METHODS: Third-six excellent condition patients were chosen and randomly divided into two groups. They were performed PKP by using fresh and cryopreserved corneas respectively. The graft transparency, epithelial healing speed of graft, tear membrane, graft thickness, endothelium density and visual acurity were emphasized respectively. RESULTS: When fresh and cryopreserved corneas were used on PKP as the donor respectively, postoperative time to recover normal break-up time (BUT) was two to six months average (4.7 +/- 0.3) months and two to eight months, average (5.6 +/- 0.4) months and time to recover normal Schirmer's test was one to four months average (2.1 +/- 0.3) months and 1-3.5 months, average (1.7 +/- 0.5) months. The graft's thickness, stabilized visual acurity and graft's rejective rate were 0.56-0.68 mm (average 0.62 mm) vs 0.54-0.62 mm (average 0.59 mm), (0.46 +/- 0.03) vs (0.44 +/- 0.05) and 20% vs 19% respectively when fresh vs cryoperserved corneas. No statistical significance between fresh and cryopreserved corneas (P > 0.05). But the grafts behaved intact epithelium and continuing transparency when fresh corneas as donors, while the graft's epithelial defects healed after three to five days of the operation and edematous grafts were reclear after two to three weeks of the operation when cryopreserved corneas as donors. Graft's endothelium density in fresh corneas group 2,043-2,210 cells/mm2 (average 2,135 cells/mm2) was high than in cryopreserved corneas 1,240-1,860 cells/mm2 (average 1,672 cells/mm2) (P < 0.01). CONCLUSION: There were no statistical significance on the transparent rate of grafts, grafts thickness, visual acurity and graft's rejective rate between fresh and cryopreserved corneas. But graft's epithelium healing was delayed and endothelium density was lower when cryopreserved corneas as donors.

Adolescent↗

Cultivation and characterization of a bovine in vitro model of the cornea.

The aim of this study was to develop an in vitro model of the cornea of bovine cells, to characterise the model by histochemical methods and to investigate permeation of ophthalmic drugs through the model. As in the in vivo situation, an in vitro model of the cornea should consist of all three different types of cells. In the current study, the construction of the in vitro cornea was performed using cells prepared from primary cultures. To investigate the state of the cells in the cultures, growth curves were established. Immunocytochemical determination of keratin and vimentin was performed for all three isolated and sub-cultivated cell types of the bovine cornea. To further simulate the in vivo conditions, corneal epithelial cells were seeded onto the collagen-gel base containing the stromal cells with an underlying sheet of endothelium. Permeation experiments were performed with pilocarpine hydrochloride and timolol hydrogen maleate as model drugs and excised bovine cornea and the in vitro cornea as permeation barriers. The immunohistochemical investigations show that excised bovine cornea and the in vitro model of the cornea are comparable with respect to the expression of keratin K3, indicating that the primarily isolated cells correspond to the different cell types of the cornea. Culturing of the epithelial cells on the complex basis has led to the formation of a corneal epithelium with several layers, closely resembling the morphology of the in vivo epithelium. Although the permeation rates of the drug through the in vitro cornea were always higher, the sequence in which the drugs permeate through the two types of barriers was the same. The drug permeation through the in vitro cornea may therefore be a useful predictive tool to estimate the permeability coefficients of drugs through excised cornea.

Adrenergic beta-Antagonists↗

Transketolase gene expression in the cornea is influenced by environmental factors and developmentally controlled events.

PURPOSE: Transketolase (TKT) has been proposed to be a corneal crystallin, and its gene and protein are abundantly expressed in the corneal epithelium of several mammals. A marked up-regulation of TKT gene expression coincides with the time of eyelid opening in the mouse. Here, we examined whether exposure to incident light contributes to the up-regulation of TKT gene expression during cornea maturation. METHODS: Mice were raised in either standard light/dark cycling conditions or total darkness. In some cases, subcutaneous injections of epidermal growth factor (EGF) were given beginning on the day of birth to induce early eyelid opening. RNA was prepared from the corneas of mothers and pups and subjected to Northern blot analyses. In addition, the relative levels of TKT mRNA and/or enzyme activity were examined in the corneas of human, bovine, rat, chicken, and zebrafish. RESULTS: TKT mRNA levels were 2.1-fold higher in the corneas of 25-day-old mouse pups ( 12 days after eyelid opening) that had been born and raised in light/dark conditions compared to pups born and raised in total darkness. By contrast, the level of TKT mRNA in the mature corneas of adult mice maintained in the dark for 2-8 weeks did not vary greatly from those of mice maintained in light/dark conditions. Interestingly, TKT mRNA levels in the corneas of dark-raised mice, although reduced, did exhibit the increase characteristically observed before and after eyelid opening. In addition, TKT mRNA levels were elevated fivefold in the corneas of 28-day-old mice raised in darkness and injected with EGF compared to uninjected mice also deprived of light. The EGF-injected mice opened their eyes 3 days early, and their corneal epithelium did not grossly differ from that of control mice. TKT mRNA and/or enzyme activity was found to be much higher in the corneas than in other tissues of humans, bovines, and rats but was extremely low in the corneas of chicken and zebrafish. CONCLUSION: Our studies suggest that both exposure to incident light and events surrounding the process of eyelid opening play a role in the up-regulation of TKT gene expression observed during corneal maturation in mice. Light appears to play a less important role in the mature cornea in maintaining high levels of TKT gene expression. The low levels of TKT in the cornea of chicken and zebrafish support the notion that TKT acts as a taxon-specific enzyme-crystallin in mammals. The involvement of environmental signals for this putative, mammalian cornea crystallin contrasts with the purely developmental signals involved in the up-regulation of the crystallin genes of the lens.

Adult↗

Graft failure in human donor corneas due to transmission of herpes simplex virus.

AIM: To report the clinical consequences of contamination of human donor corneas by herpes simplex virus (HSV) in organ culture. METHODS: Two patients without previous history of ocular HSV infection underwent penetrating keratoplasty (PK), one for keratoconus and the other for Fuchs' endothelial dystrophy. One patient suffered primary graft failure while the other developed a persistent epithelial defect, ultimately resulting in graft failure. Viral culture of swabs taken from both corneas during the early postoperative period was undertaken. The failed donor corneas were examined histopathologically by immunohistochemistry (IHC) for HSV-1 antigens, transmission electron microscopy (TEM), and by polymerase chain reaction (PCR) for HSV DNA. Both failed corneas were replaced within 6 weeks of the initial surgery. The records of the fellow donor corneas were also examined for evidence of infection. RESULTS: HSV was cultured from both corneas during the early postoperative period. Histology of both donor corneas demonstrated a thickened corneal stroma with widespread necrosis of keratocytes and loss of endothelial cells. IHC showed keratocytes positive with antibodies to HSV-1 antigens. TEM demonstrated HSV-like viral particles within degenerating keratocytes. PCR performed on the failed corneal grafts was positive for HSV-1 DNA, whereas PCR performed on the excised host corneal buttons was negative in both patients. Records of the fellow donor corneas showed that one cornea was successfully transplanted into another recipient after 18 days in organ culture, whilst the other was discarded because of extensive endothelial cell necrosis noted after 15 days in organ culture. CONCLUSION: HSV within a donor cornea may cause endothelial destruction in organ culture and both primary graft failure and ulcerative keratitis after transplantation. Endothelial necrosis of a donor cornea in culture also raises the possibility of HSV infection within the fellow cornea.

Adult↗

Dynamics of the expression of cytoskeleton components and adherens molecules by fibroblastic cells in alkali-burned and lacerated corneas.

To provide a better understanding of the role of fibroblastic cells during corneal wound-healing, we examined the expression of cytoskeleton components (i.e. smooth muscle alpha-actin (alpha-SMA), vimentin, desmin), adherens molecules (vinculin and talin) and cellular fibronectin in alkali-burned and lacerated rabbit corneas. Alkali-burned and lacerated corneas, which had healed for various periods of time (1 day to 45 days), were excised and subjected to immunohistochemical studies with monoclonal antibodies against alpha-SMA, vimentin, desmin, vinculin, talin and cellular fibronectin. Monoclonal antibody against alpha-SMA reacted with fibroblastic cells in injured corneas but did not react with keratocytes in normal corneas. Anti-desmin antibody did not react with any corneal cells in normal or injured corneas except the muscle cells in the newly-formed capillary of injured corneas. The results indicate that the fibroblastic cells in injured corneas have the characteristics of myofibroblasts. The number of myofibroblasts in granulation tissues increased and peaked within 3 weeks of injury and then declined. Electron microscopy revealed that some fibroblastic cells in the lacerated cornea which had healed for 4 weeks contained dense chromatins. In situ 3'-end labeling with terminal nucleotide transferase indicating that some of the fibroblastic cells contained nicked genomic DNA. These observations imply that apoptosis plays a role in regulating the number of myofibroblasts in the injured corneas. Antibodies against cellular fibronectin, vinculin and talin react with the fibroblastic cells in the injured corneas, but not with the keratocytes of normal corneas. Examination with transmission electron microscopy demonstrated the presence of microtendon and fibronexis associated with fibroblastic cells and the presence of stress fiber within fibroblastic cells. The results indicate that the fibroblastic cells may cause corneal wound contraction, which in turn contributes to the formation of opaque scar tissues.

Animals↗

Cornea-lens transdifferentiation in the anuran, Xenopus tropicalis.

Previously, the only anuran amphibian known to regenerate the lens of the eye was Xenopus laevis. This occurs during larval stages through transdifferentiation of the outer cornea epithelium under control of factors presumably secreted by the neural retina. This study demonstrates that a distantly related species, X. tropicalis, is also able to regenerate lenses through this process. A transgenic line of X. tropicalis was used to examine the process of cornea-lens transdifferentiation in which green fluorescent protein (GFP) is expressed in differentiated lens cells under the control of the Xenopus gamma1-crystallin promoter element. Unlike X. laevis, the process of cornea-lens transdifferentiation typically occurs at a very low frequency in X. tropicalis due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening. The inner cornea endothelium serves as a key physical barrier that normally prevents retinal signals from reaching the outer cornea epithelium. If this barrier is circumvented by implanting outer cornea epithelium of transgenic tadpoles directly into the vitreous chamber of non-transgenic X. tropicalis larval eyes, a higher percentage of cases formed lenses expressing GFP. Lenses were also formed if these tissues were implanted into X. laevis larval eyes, suggesting the same or similar inducing factors are present in both species. When pericorneal ectoderm and posteriolateral flank ectoderm were implanted into the vitreous chamber, only in rare cases did pericorneal ectoderm form lens cells. Thus, unlike the case in X. laevis, competence to respond to the inducing factors is tightly restricted to the cornea epithelium in X. tropicalis. As controls, all these tissues were implanted into the space located between the inner and outer corneas. None of these implants, including outer cornea epithelium, exhibited GFP expression. Thus, the essential inductive factors are normally contained within the vitreous chamber. One explanation why this type of lens regeneration is not seen in some other anurans could be due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening once the original lens is removed. These findings are discussed in terms of the evolution of this developmental process within the anurans.

Animals↗

Cornea fluid dynamics. I: measurement of hydrostatic and colloid osmotic pressure in rabbits.

Interstitial fluid pressure (Pi) has been measured in vivo in ocular tissues of rats and rabbits, and in vitro in corneas of cows and humans with micropipettes connected to a servocontrolled counterpressure system. The in vivo measurements were performed after anaesthesia and careful immobilization of the head. In all species Pi became gradually more negative from the limbus to the cornea centre; in rabbits the change was from -17.8 mmHg 0.5 mm centrally to the limbus to -42.7 mmHg in central cornea. Cornea and sclera interstitial fluid was isolated by centrifugation, and was used for analysis of colloid osmotic pressure (both tissues) and Na(+)-concentration and electrophoresis (cornea only). During low speed centrifugation (3000 rpm), a wet weight fraction of 2-4% was isolated from cornea, having a colloid osmotic pressure not significantly different from that of plasma (18.9 mmHg). Na+ analysis suggested that the isolated fluid did not derive from corneal cells. Electrophoresis showed a protein pattern in cornea fluid similar to that of plasma except for a band with MW corresponding to 39,000 found in cornea only. The present study demonstrates a pressure gradient from sclera to cornea, and along cornea from periphery to centre, and suggests that proteins in cornea fluid contribute significantly to swelling pressure.

Animals↗

A comparison of biomechanical properties between human and porcine cornea.

Due to the difficulty in obtaining human corneas, pig corneas are often substituted as models for cornea research. The purpose of this study is to find the similarities and differences in the biomechanical properties between human and porcine corneas. Uniaxial tests were conducted using an Instron apparatus to determine their tensile strength, stress-strain relationship, and stress-relaxation properties. The tensile strength and stress-strain relation were very similar but significant differences between the two tissues were observed in the stress-relaxation relationship. Under the same stretch ratio lambda=1.5, porcine cornea relaxed much more than human cornea. If tensile strength and the stress-strain relation are the only mechanical factors to be investigated, porcine cornea can be used as a substitute model for human cornea research. However, when stress relaxation is a factor, porcine corneas cannot be used as an appropriate model for human corneas in mechanical property studies. It is very difficult to get enough specimens of human cornea, so we did the experiments for stress-strain relationship at a specific value of strain rate (corresponding to the velocity of loading 10mm/min), and for stress relaxation at a specific stretch ratio lambda=1.5.

Animals↗